Distribution of Average Aggregate Density from Stir-Stressed NISTmAb Protein

Quantifying the heterogeneous nature of protein aggregates is important to understanding the impact aggregates may have on the performance of antibody therapeutics. The spatially averaged density ρ_p of aggregates, defined as the total mass, including water, divided by the volume, is a parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of aggregated protein in a sample. We report measurements by two methods on the distribution of density values for different aggregate sizes, where the aggregates were produced by stir-stressing fluorescently labeled monoclonal antibody (NISTmAb). A fluorescence microscope was used to image particles. Each particle was analyzed for brightfield equivalent circular diameter (ECD) and fluorescence intensity and the results converted to average density. Measurements were also obtained using video holography. The aggregates were highly porous with median density decreasing from 1.07 g/cm³ to 1.02 g/cm³ as the size increased from 0.9 μm to 6 μm by fluorescence, and similar results by video holography. The distribution in density for a given particle size was asymmetrical and broad. For example, particles with an ECD of 2.5 μm ranged in density from 1.005 g/cm³ to 1.1 g/cm³.     

Quantitative Laser Diffraction for Quantification of Protein Aggregates: Comparison With Resonant Mass Measurement,Nanoparticle Tracking Analysis,Flow Imaging,and Light Obscuration

In the past, analysis of micron-sized (>1.0 μm) aggregates of therapeutic proteins has been limited to light obscuration (LO), and appropriate quantitative methods of evaluating protein aggregates need to be developed. Recently, novel methods with enhanced reliability and sensitivity, such as nanoparticle tracking analysis (NTA), resonant mass measurement (RMM), and flow imaging (FI), have emerged. We have found that quantitative laser diffraction (qLD) is also effective for quantitative evaluation of protein aggregates over a wide size range. However, the different detection principles of the methods potentially lead to inconsistencies in results. This study aimed to compare particle size distributions and concentrations of protein aggregates using the orthogonal methods. Protein aggregates were generated by stirring an immunoglobulin solution. Serial dilutions of the aggregates stock were analyzed by RMM, FI, and qLD to obtain the particle size distribution and concentration using each method. In addition, size distribution of a protein aggregates solution was compared by RMM, NTA, FI, LO, and qLD. Both particle size distribution and concentration were in good agreement between RMM and qLD (0.3-2 μm) and between FI and qLD (2-20 μm). Thus, we concluded that qLD enables covering of the overlapping particle size range between RMM and FI.     

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches

Purpose

Characterizing submicron protein particles (approximately 0.1–1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS).

Methods

Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment.

Results

A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4?×?10⁷-1?×?10¹¹ particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering.

Conclusion

RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

     

Flixotide™‐pressurized metered‐dose inhalers loaded with [18F]fluticasone propionate particles for drug deposition studies in humans with PET–formulation and analysis

Fluticasone propionate (FP) is a potent anti‐inflammatory synthetic steroid, used for the treatment of asthma. Flixotide? is a formulated pressurized metered‐dose inhaler (pMDI) that contains small‐micronized FP particles in a blend of CFC propellants. Our objective was to develop a radiotracer method for accurately measuring the regional deposition of FP within the human lung using positron emission tomography (PET), which would be of important clinical interest. Flixotide? pMDIs were used to prepare [¹⁸F]FP pMDIs labeled isotopically with the positron emitter, fluorine‐18 (t_1/2=109.7 min). FP particles from Flixotide? pMDIs were mixed with [¹⁸F]FP formulated into a pMDI and sonicated at room temperature. The drug delivery of [¹⁸F]FP pMDI (250 μg of FP per actuation dose) was assessed for particle size distribution and dose uniformity. The distributions of FP and [¹⁸F]FP across particle size in such preparations were measured with an Andersen cascade impactor. This procedure was shown to provide an emitted dose from a [¹⁸F]FP pMDI of 246±19 μg/per metered dose. The particle size distribution as measured by mass median aerodynamic diameter (MMAD) (The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) for each distribution were calculated. MMAD is defined as the aerodynamic diameter around which the mass of particles is equally distributed and the GSD is a measure of the dispersion of these particle diameters around the MMAD) from a commercial Flixotide? pMDI was 2.6±0.2 μm and agreed well with that from an [¹⁸F]FP pMDI (2.8±0.1 μm). The MMAD and geometric standard deviation (GSD) of newly formulated [¹⁸F]FP pMDIs were unaffected by the formulation procedure. [¹⁸F]FP was distributed with good uniformity with respect to the mass of FP for particles greater than 0.43 μm. Hence, the radiolabeled pMDI is a suitable source of radiotracer for the regional measurement of lung deposition for inhaled FP in human subjects with PET. Copyright © 2003 John Wiley & Sons, Ltd.     

An infrared spectrophotometric study of the compaction mechanism of potassium bromide

Potassium bromide has been compacted in a vacuum die at pressures up to 1000 MN m^?2, and the infrared transmittance of the resulting flat discs measured. Four mean particle sizes were used: 71, 110, 388 and 550 μm. Over the whole range of pressure, the 110 μm material had a higher transmittance. At any one particle size, the transmittance increased with increasing compaction pressure up to 400 MN m^?2, then fell slightly and was followed by a less pronounced increase in the region of 1000 MN m^?2. The following mechanical properties of the compacts were also investigated: tensile strength, relative density, surface hardness and dissolution time. The transmittance changes have been related to the probable compaction mechanism.     

Estimation of Drug Particle Size in Intact Tablets by 2-Dimensional X-Ray Diffractometry

The average grain size of a crystalline material can be determined from the γ-profile of Debye rings in 2-dimensional X-ray diffraction frames. Our objectives were to: (1) validate the method for organic powders and use it to determine the grain size in intact tablets, and (2) demonstrate the pharmaceutical application of this technique by determining the grain size of the active pharmaceutical ingredient in marketed formulations. Six sieve fractions of sucrose were prepared and the particle size distribution was confirmed by laser diffraction. Their average grain size was determined from the 2-dimensional X-ray diffraction frames by the γ-profile method. For particles <90 μm (based on sieving), the average particle size determined by the 3 methods were in good agreement. When these particles were compressed, there was no discernible change in the sucrose grain size in tablets. When the particles were >250 μm, compression resulted in a mixture of large grains and fine powder. The grain size of acetaminophen in 11 marketed tablet formulations was determined to be either ～35 μm or ～80 μm. This nondestructive technique can therefore be potentially useful to estimate the grain size of crystalline formulation components in intact tablets.     

Particle Size and Charge Distribution Analysis of Pharmaceutical Aerosols Generated by Inhalers

Aerosol particles generated by inhalers for respiratory drug delivery acquire electrostatic charge during the dispersion process. The electrostatic charge distribution of the particles can affect the efficiency of drug delivery by influencing both the transport and deposition of inhaled particles in the human lung. To analyze the electrostatic charge acquired by the aerosol particles, two sets of metered-dose inhaler (MDI) and dry powder inhaler (DPI) devices were investigated. Both the particle size and charge distributions were measured simultaneously by using an electrical single-particle aerodynamic relaxation time (E-SPART) analyzer. The analyzer was calibrated with particles of known size, which were generated by a vibrating orifice aerosol generator (TSI Inc.) and charge using the Faraday cup method. The charge distributions of the pharmaceutical aerosols from both the DPI and MDI devices were bipolar in nature. Although the net charge-to-mass ratio was less than 0.2 μC/g, the individual particles were charged with a relatively high charge: ?2 to + 2 μC/g. The count mean aerodynamic diameter of the aerosols generated from these devices was 3–5 μm.     

High-Output Generation of Aerosols with Narrow Size Distributions

Abstract

Two aerosol generators–a small particle generator (SPG) and a large particle generator (LPG)–were designed and fabricated to produce water-soluble particles with high mass output and narrow size distributions. The mass median aerodynamic diameter (MMAD) of solid particles produced could be varied by changing operation conditions and using different concentrations of sodium chloride (NaCl) solutions. The aerosol generation rate varied from about 0.2 to 24 mg/min depending upon the particle size produced as the geometric standard deviation (GSD) was maintained below 1.5. The SPG employed a Collison-type nebulizer with multiple nozzles and a solid-plate impactor, which removes generating droplets larger than the cutpoint diameter for the production of submicrometer aerosols with GSD < 1.5. Different combinations of nebulization pressure/cutpoint diameter were selected to produce solid particles with MMAD in the range of 0.13–1.0 μm. The LPG was consisted of a Delavan simplex nozzle installed at the bottom of the generation chamber (about 190 cm in height and 15 cm in diameter) and an improved virtual impactor located at the top of the generation chamber. Gravity was used to remove large droplets and the improved virtual impactor was employed to remove droplets less than the cutpoint diameter. Two sets of acceleration nozzle and collection probe were used to vary the impactor cut size. The size-selective droplets were then evaporated to form solid particles with the MMAD nominally varying from 1 to 10 μm and GSD < 1.5.     

Aerodynamic size distribution,hygroscopicity and deposition estimation of beclomethasone dipropionate aerosol

Aerodynamic size distribution and aerodynamic mass per dose of beclomethasone dipropionate aerosol were measured at 24 and 95% relative humidity. At high humidity, the count median aerodynamic diameter was unchanged, mass median aerodynamic diameter increased from 2·01 μm to 2·68 μm, particle number/dose from 41·3 times 10⁶ to 78·3 times 10⁶, and aerodynamic mass per dose from 23·7 to 60·0μ g. The quantity of active ingredient estimated to be in the 23·7μg aerodynamic mass at low humidity was 19·7 μg. From data previously available describing average deposition fraction as a function of aerodynamic diameter, 6·7 μg or 13% of the total dose of 50μg produced by the metered dose canister would be expected to deposit in the lower respiratory tract.     

Particle size distribution of heavy metals in the urban air of Seoul,Korea

Atmospheric particulate matters (A.P.M.) were collected on quartz-fiber filters from March 1985 to May 1986, using the Andersen high-volume air sampler and contents of six heavy metals (Fe, Mn, Cu, Zn, Pb, Ni) in the A.P.M. were determined by atomic absorption spectrophotometry. These heavy metals were divided into the three groups with respect to their particle size distribution. Fe and Mn were mainly associated with coarse particles (diameter >2.0 μm), but Pb and Ni were related fine particles (diameter <2.0 μm). Cu and Zn had mixed size distributions in both of them. In the seasonal variation of heavy metals, the contents of Fe and Mn in spring and Ni and Pb in winter were higher than any other season. There were high mutual correlation between Fe and Mn in coarse particles, and between Pb and Ni in fine particles.     

Characterization of particles in protein solutions: reaching the limits of current technologies

Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2-10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5-150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.     

The effects of substrate size, surface area, and density on coat thickness of multi-particulate dosage forms

Drugs layering experiments were performed in a fluid bed fitted with a rotor granulator insert using diltiazem as a model drug. The drug was applied in various quantities to sugar spheres of different mesh sizes to give a series of drug-layered sugar spheres (cores) of different potency, size, and weight per particle. The drug presence lowered the bulk density of the cores in proportion to the quantity of added drug. Polymer coating of each core lot was performed in a fluid bed fitted with a Wurster insert. A series of polymer-coated cores (pellets) was removed from each coating experiment. The mean diameter of each core and each pellet sample was determined by image analysis. The rate of change of diameter on polymer addition was determined for each starting size of core and compared to calculated values. The core diameter was displaced from the line of best fit through the pellet diameter data. Cores of different potency with the same size distribution were made by layering increasing quantities of drug onto sugar spheres of decreasing mesh size. Equal quantities of polymer were applied to the same-sized core lots and coat thickness was measured. Weight/weight calculations predict equal coat thickness under these conditions, but measurable differences were found. Simple corrections to core charge weight in the Wurster insert were successfully used to manufacture pellets having the same coat thickness. The sensitivity of the image analysis technique in measuring particle size distributions (PSDs) was demonstrated by measuring a displacement in PSD after addition of 0.5% w/w talc to a pellet sample.     

On the Production of Chitosan-Coated Polycaprolactone Nanoparticles in a Confined Impinging Jet Reactor

This work is focused on the synthesis of polycaprolactone nanoparticles, coated with chitosan, in a confined impinging jet reactor using the solvent displacement method. The role of the various reacting species was investigated, evidencing that a biocompatible polymer, for example, polycaprolactone, is required to support chitosan to obtain a monomodal particle size distribution, with low particle diameters. A surfactant is required to reduce the nanoparticle size (down to a mean diameter of about 260 nm) and obtain a positive zeta potential (about +31 mV), perfectly suitable for pharmaceutical applications. Different surfactants were tested, and Poloxamer 388 appeared to be preferable to polyvinyl alcohol. The effect of the concentration of Poloxamer 388 (in the range 0.5-5 mg mL^?1) and of chitosan (in the range 1.5-5 mg mL^?1) on both the mean particle size and zeta potential was also investigated, evidencing that chitosan concentration has the strongest effect on both parameters. Finally, the effect of solvent evaporation, quenching and feed flow rate was investigated, showing that the evaporation stage does not affect particle characteristics, quenching is required to avoid particle aggregation, and a minimum liquid flow rate of 80 mL min^?1 is required in the considered reactor to minimize the particle size.     

Mean particle diameters. From statistical definition to physical understanding

Mean particle diameters may be used to describe and to model physical, chemical, or physiological properties of products or materials containing dispersed phases. There are different notation systems for these mean diameters, which may cause much confusion. This equally applies to their nomenclature. This article introduces the Moment-Ratio definition system and evaluates briefly the ISO definition system. The ISO system appears to have serious drawbacks. Mean particle diameters can be estimated from histograms of size distributions by Summation (M-R system) and by Integration (ISO system) over the histogram intervals. Summation tends to be more accurate than Integration and is less sensitive to low values of the lower limit of size distributions. The Summation method equations are straightforward and generally applicable. The mathematical formulas of the Integration method are difficult to apply in daily practice, and their complexity may easily hide the physical background of a mean particle diameter. A coherent nomenclature system for denoting mean particle diameters is recommended. This nomenclature system does not contain any ambiguities and clearly conveys the physical meanings of mean particle diameters. This article deals also with an empirical method to select the proper type of mean diameter to describe a physical, chemical, or physiological property of a product or material containing dispersed phases. After calculation of the mean diameters from experimental data, the relationships between the product property and these mean diameters are investigated statistically. The selection method has been illustrated by two examples. The dataset of each example consists of a set of particle size distributions and the corresponding physical product properties that are influenced by the particle sizes. Hypotheses are formulated to explain the types of selected mean diameters. Sharing results from all over the world of applications of the developed selection method will lead to a buildup of knowledge of physical meanings and application areas of the types of mean particle diameters, which will support decision making in product development.     

Kinetics of the sorption of tetracycline by anion exchanger AB-17-2 with different dispersions

The kinetics of tetracycline sorption on the anion exchanger AB-17-2 were found to show a significant dependence on exchanger particle size. Experimental studies demonstrated the transition from gel to mixed and film diffusion during tetracycline sorption when anion exchanger particle size decreased from 800–1200 μm to 0.1–1.0 μm, while the coefficients of diffusion were comparable for model and real solutions, varying from 10¹⁰ to 10⁷ cm²/sec. These data demonstrate potential for the use of anion exchanger AB-17-2 in the extraction and purification of tetracycline. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 41, No. 8, pp. 39–41, August, 2007.     

Use of Analytically Defined Estimates of Aerosol Respirable Fraction to Predict Lung Deposition Patterns

Analytical estimates of the respirable fractions on inhaled pharmaceutical aerosols are obtained by inertial sampling techniques. The respirable fraction may be defined as that portion of the particle size distribution less than a designated diameter. The diameter size below which particles were considered respirable in these studies was 6.4 µm. In clinical practice, a variety of particle size distributions may be related to a single respirable fraction. Herein, three respirable fractions were each defined by six particle size distributions. The deposition patterns of aerosols exhibiting these particle size characteristics were examined in a mathematical model. The analytically defined respirable fractions were compared with predicted lung deposition values. Under clearly defined breathing conditions, there is a correlation between the nominal respirable fraction and deposition. However, it was concluded that the variations which occur in breathing parameters within patient populations may not allow a single analytically derived respirable fraction to be appropriate for all individual subjects.     

The vibratory consolidation of particle size fractions of powders

The use of controlled sinusoidal vibration as a means of consolidating packings of lactose within small containers has been examined. Vertical vibration was found significantly more effective and reproducible than horizontal vibration in terms of the degree of consolidation achieved. An optimum frequency range was identified within which the densification was greatest, and this range was largely independent of particle size for particle size fractions of mean volume diameters ranging from 15.6 to 155 μm. The consolidation increased with increasing vibration acceleration up to a level beyond which no further decrease in porosity resulted. Typical effective vibration conditions were characterized by amplitudes of an order of magnitude similar to the particle sizes studied. For particle size fractions of mean diameters 17·8, 37·5 and 80·8 μm, there is evidence that an optimum particle size range exists, within which energy requirements for consolidation are at a minimum.     

The optimal particle size for parasympathicolytic aerosols in mild asthmatics

Background: the optimal particle size of a parasympathicolytic aerosol is unknown. Methods: eight stable asthmatics with a mean FEV₁ of 72% of the predicted value inhaled three types of monodisperse ipratropium bromide aerosols, with particle sizes of 1.5, 2.8 and 5 μm, respectively, and a placebo aerosol. The volunteers inhaled 8 μg ipratropium bromide, after which lung function improvement was determined. The changes in lung function were analysed with repeated measurements ANOVA. Results: according to the changes in FEV₁ and MEF_50/25 the 1.5/2.8 μm aerosol induced significantly better bronchodilatation than the 5 μm aerosol. No particle size effect was noticeable with regard to changes in R_cot, VC, FVC and PEF. Conclusions: in mild asthmatics the particle size of choice for a parasympathicolytic aerosol should be ≤ 2.8 μm.     

Mixing of a mAb Formulation in a New Magnetically Coupled Single-Use Mixing System: Key Learnings of Preliminary Experimental and Computational Evaluation

MilliporeSigma recently introduced a new magnetically coupled single-use mixing system (Mobius® Power MIX) for more efficient mixing of buffers and media in biopharmaceutical applications. Experimental and computational fluid dynamics (CFD) assessments were performed on the Power MIX 100 system to understand product quality impact, shear, and mixing efficiency. It was interesting to note slightly higher submicron (0.4-1 μm) and subvisible (1-54 μm) particle formation at the lower mixing speed (50 RPM) compared to higher mixing speeds (100/200 RPM). Mixing speed and time showed negligible impact on the other product quality attributes tested, including protein concentration, turbidity, general appearance, purity, and soluble aggregates. The CFD simulations provided useful information with respect to the impact of batch size (20-100 L), viscosity (2-50 cP), and impeller speed (100-300 RPM) on mixing time (mixing time ranged from 10 to 365 s) and shear (maximum shear rate was found to be localized around the impeller and it was about 30,260 s^?1, whereas the average shear rate ranged from 4 to 36 s^?1). Statistical analysis of the CFD results showed that natural-log transformation and quadratic fitting were found to be suitable statistical models to predict mixing time and shear within the design space of the parameters assessed in the present study.     

Sustained release indomethacin-loaded solid lipid microparticles based on solidified reverse micellar solution (SRMS): in vitro and in vivo evaluation

Solidified reverse micellar solutions (SRMS) were formulated using phospholipid (Phospholipon 90H) and triglyceride (Softisan 154) in the ratios of 1:1, 2:1 and 1:2, respectively. SLMs were prepared by melt homogenization technique. Characterization based on yield, mean particle diameter and morphology, pH, thermal analysis and encapsulation efficiency (EE%) were carried out on the SLMs. In vitro release was carried out in simulated intestinal fluid (SIF, pH 7.5), while the anti-inflammatory and ulcerogenic properties were studied using rats. From the results, the mean particle diameter of SLMs ranged from 2.19 ± 0.05 μm to 20.77 ± 0.03 μm. Maximum EE% of 96, 93 and 94 % were obtained and showed significant variation within the batches (p < 0.05). The release profile of indomethacin-loaded SLMs showed about 82-99 % drug release at 13 h. Indomethacin-loaded SLMs showed good anti-inflammatory and gastro-protective effects, and could be formulated for once-daily administration, either orally or parenterally, under controlled conditions.